The long term objective is to understand the cellular and molecular basis for neuronal C - homeostasis. Intracellular [CI-] ([Cl-]i) of neurons is important because it is a key factor in determining the Cl-electrochemical gradient across the plasma membrane and therefore dictates the response (i.e., depolarizing vs. hyperpolarizing) of ligand-gated anion channels, like GABA-A receptors (GABAARs). While fast GABAergic transmission mediated by GABAARs is predominantly hyperpolarizing and inhibitory in the adult, it is not static and undergoes remarkable shifts in polarity in both physiology and pathophysiology. We hypothesize that such polarity shifts in the voltage response of the GABAAR are the direct result of changes in the transport capacities of the major neuronal "Cl- pumps", i.e., the Na-K-CI cotransporter (NKCC) and K-CI cotransporter (KCC). We have identified and characterized a neuron-specific isoform of the K-CI cotransporter (KCC2) that functions primarily as a Cl- extrusion mechanism. This proposal has four specific aims; 1) To elucidate the operation of KCC2. We will test a functional model of KCC2 as a "dynamic buffer" of neuronal [CI-] and external [K+], determine how changes in [Cl-]i modulate KCC2 activity, and test a kinetic model for KCC2. 2) To characterize ammonium transport by KCC2. Preliminary studies show that NH4+ is translocated by KCC2. We will use NH4+ translocation to characterize KCC2 activity in individual cultured neurons using pH-sensitive fluorescent dyes. 3) To elucidate the molecular mechanism of acute regulation of KCC2. We hypothesize that acute regulation of KCC2 involves changes in its phosphorylation state. We will address this hypothesis using KCC2 protein in both native and heterologous expression systems. 4) To determine the role of membrane trafficking in regulating KCC2 transport capacity. Using both native and heterologous expression systems, we will correlate changes in surface KCC2 density with transporter activity. Our results will provide a rational basis for understanding neuronal Cl- homeostasis as well as the cellular and molecular mechanisms responsible for polarity shifts in fast GABAergic transmission observed in physiology and pathophysiology.